Abstract
We recently reported that trace insertion of exogenous fluorescent (green BODIPY) analogs of sphingomyelin (SM) into living red blood cells (RBCs), partially spread onto coverslips, labels submicrometric domains, visible by confocal microscopy. We here extend this feature to endogenous SM, upon binding of a SM-specific nontoxic (NT) fragment of the earthworm toxin, lysenin, fused to the red monomeric fluorescent protein, mCherry [construct named His-mCherry-NT-lysenin (lysenin*)]. Specificity of lysenin* binding was verified with composition-defined liposomes and by loss of (125)I-lysenin* binding to erythrocytes upon SM depletion by SMase. The (125)I-lysenin* binding isotherm indicated saturation at 3.5 × 10(6) molecules/RBC, i.e., ∼3% of SM coverage. Nonsaturating lysenin* concentration also labeled sub-micrometric domains on the plasma membrane of partially spread erythrocytes, colocalizing with inserted green BODIPY-SM, and abrogated by SMase. Lysenin*-labeled domains were stable in time and space and were regulated by temperature and cholesterol. The abundance, size, positioning, and segregation of lysenin*-labeled domains from other lipids (BODIPY-phosphatidylcholine or -glycosphingolipids) depended on membrane tension. Similar lysenin*-labeled domains were evidenced in RBCs gently suspended in 3D-gel. Taken together, these data demonstrate submicrometric compartmentation of endogenous SM at the membrane of a living cell in vitro, and suggest it may be a genuine feature of erythrocytes in vivo.
Highlights
We recently reported that trace insertion of exogenous fluorescent analogs of sphingomyelin (SM) into living red blood cells (RBCs), partially spread onto coverslips, labels submicrometric domains, visible by confocal microscopy
We previously reported that insertion at trace level into the outer plasma membrane leaflet of exogenous SM, modified by replacing part of its fatty acyl chain by the green fluorophore BODIPY (BODIPY-SM), labels submicrometric assemblies visible by confocal microscopy on various cells, including cultured cell lines and red blood cells (RBCs) partially spread onto poly-L-lysine-coated coverslips [9,10,11]
Because we had previously shown that temperature and cholesterol regulated the abundance of BODIPY-SM domains [9,10,11], we investigated whether they likewise affected endogenous SM submicrometric domains labeled by lysenin*
Summary
We recently reported that trace insertion of exogenous fluorescent (green BODIPY) analogs of sphingomyelin (SM) into living red blood cells (RBCs), partially spread onto coverslips, labels submicrometric domains, visible by confocal microscopy. (20), and further indicated by single-molecule tracking, based on discrete jumps between predicted mesoscale domains [6] Besides this dynamic evidence, we previously reported that insertion at trace level into the outer plasma membrane leaflet of exogenous SM, modified by replacing part of its fatty acyl chain by the green fluorophore BODIPY (BODIPY-SM), labels submicrometric assemblies visible by confocal microscopy on various cells, including cultured cell lines and red blood cells (RBCs) partially spread onto poly-L-lysine-coated coverslips [9,10,11]. Domains of similar size have been reported for yeast plasma membrane proteins [24, 25]
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